The novel substrates are useful as reagents in the detection and determination of proteolytic enzymes which cleave amide linkages in peptide chains on the carboxyl side of arginine and lysine residues.
Structurally related chromogenic and fluorometric substrates have been described in U.S. Pat. Nos. 3,886,136; 4,028,318; 4,061,625 and 4,070,245; however, none of these substrates benefit from substituting an .alpha. hydroxide for an .alpha.-amino group in the terminal moiety.
Specifically, it has been found that when an analogous .alpha. hydroxy acid is substituted at the N terminous or P.sup.3 site of a tripeptide substrate at least three advantages are realized. Most notably, solubility in aqueous buffers is greater with the .alpha. hydroxy than with benzoyl blocked amino acid substrates. In addition, the .alpha. hydroxy substrates show no susceptibility to amino peptidase activity thereby eliminating the need for blocking groups on the N terminous and obviating the use of the D form over the L form (natural) of the N terminous amino acid. The .alpha. hydroxy analogs also simplify synthesis by eliminating the need to block and deblock the hydroxy substituent during synthesis. The .alpha. hydroxy substrates also show similar enzymatic reactivities with kinetics very similar to their .alpha. amino acid counterparts.